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A Novel Highly Sensitive Method for Measuring Inflammatory Neural-Derived APC Activity in Glial Cell Lines, Mouse Brain and Human CSF.

International journal of molecular sciences (2020-04-05)
Valery Golderman, Shany G Gofrit, Nicola Maggio, Orna Gera, Alexandra Gerasimov, Dar Laks, Joab Chapman, Efrat Shavit-Stein
ABSTRACT

Neural inflammation is linked to coagulation. Low levels of thrombin have a neuroprotective effect, mediated by activated protein C (APC). We describe a sensitive novel method for the measurement of APC activity at the low concentrations found in neural tissue. APC activity was measured using a fluorogenic substrate, Pyr-Pro-Arg-AMC, cleaved preferentially by APC. Selectivity was assessed using specific inhibitors and activators. APC levels were measured in human plasma, in glia cell lines, in mice brain slices following mild traumatic brain injury (mTBI) and systemic lipopolysaccharide (LPS) injection, and in cerebrospinal fluid (CSF) taken from viral meningoencephalitis patients and controls. Selectivity required apixaban and alpha-naphthylsulphonylglycyl-4-amidinophenylalanine piperidine (NAPAP). APC levels were easily measurable in plasma and were significantly increased by Protac and CaCl2. APC activity was significantly higher in the microglial compared to astrocytic cell line and specifically lowered by LPS. Brain APC levels were higher in posterior regions and increased by mTBI and LPS. Highly elevated APC activity was measured in viral meningoencephalitis patients CSF. This method is selective and sensitive for the measurement of APC activity that significantly changes during inflammation in cell lines, animal models and human CSF.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥98.5% (GC)
Sigma-Aldrich
Thrombin from bovine plasma, lyophilized powder, 40-300 NIH units/mg protein (biuret)
Sigma-Aldrich
Sodium orthovanadate, ≥90% (titration)
Millipore
Protease Inhibitor Cocktail Set III, EDTA-Free - Calbiochem, Protease inhibitor cocktail III, EDTA-free for inhibiting aspartic, cysteine, and serine proteases as well as aminopeptidases in mammalian cells and tissues.
Sigma-Aldrich
Lipopolysaccharides from Escherichia coli O111:B4, purified by trichloroacetic acid extraction
Sigma-Aldrich
Octoclothepin maleate salt, solid
BRAND® 96-well microplate, U-bottom, round bottom, non-sterile